Genetic linkage studies have demonstrated that there are two forms of dominant polycystic kidney disease (ADPKD): a form (PKD1) in which the mutation is linked to alpha-globin on chromosome 16 and a form which is not linked (PKD2). The long-term aim of this proposal is to isolate the gene(s) mutated in the unlinked form of ADPKD as a basis for studying the pathogenesis of PKD2. Since the biochemical defect in ADPKD is not known, the 'reverse genetic' approach will be used to clone the PKD2 gene(s). PKD2 will be localized by genetic linkage studies using randomly-selected highly polymorphic DNA probes from all autosomes. Additional markers will be used to define a genetic interval containing the disease gene. Infrequent-cutter linking libraries will be used in combination with in situ hybridization to provide new markers from the region of interest. These markers will be physically linked by pulsed-field electrophoresis of infrequent-cutting restriction enzyme fragments. The restriction map so generated will be used to guide the cloning of the entire PKD2 region into yeast artificial chromosomes and overlapping cosmids. These clones will be used as probes to isolate cDNAs from the region and to detect restriction fragment alterations generated by disease munitions. Denaturing gradient gel electrophoresis will also be used to detect disease-related mutations. The cloned PKD2 gene will be used to study the genomic organization of PKD2, to characterize PKD2 mutations and to correlate the genotype with the phenotype. In the long term, the cloned PKD2 gene will allow the PKD2-coded peptide to be studied and its relationship to the P3M1 product to be determined.